SUMMARY: Methamphetamine (METH) abuse can cause neuronal apoptosis, resulting in a spectrum of mild to moderate neurocognitive disorders that may progress to dementia. Neurocognitive disorders are a result of damage to the nerve terminals of dopamine-producing neuronal cells that trigger activation of apoptotic mechanisms, causing complete disintegration and death of neurons, astroglia and microglia. Chronic use of METH, an addictive psychostimulant, produces neurotoxicity by causing microglial activation and resultant secretion of pro-inflammatory cytokines, leading to neural injury and cell death, or apoptosis. Our main hypothesis is that METH treatment significantly impacts mitochondrial respiration and induces activation of the mitochondrial-dependent intrinsic apoptotic pathway, resulting in neuronal apoptosis. This project utilizes innovative optics-based methodologies, such as three dimensional quantitative phase imaging and Raman spectroscopy, to study Methamphetamine (METH) -induced cell apoptosis in primary neurons in real time. By monitoring cell apoptosis in human neurons non-invasively, in real time using quantitative phase imaging (QPI), we will observe morphological changes in cells. For QPI, we will use both digital holographic microscopy (DHM) and transport of intensity imaging (TIE). TIE is a relatively new QPI method in microscopy, which offers some significant advantages over the better-developed DHM methods, including compatibility at low cost with existing bright-field microscopes, greater stability and robustness to vibration and improved spatial resolution. A novel TIE QPI system will be developed as part of the proposed work. Further, by monitoring their Raman spectroscopic signatures during various stages of cell apoptosis, we will detect chemical changes/protein content within neurons. We will examine METH induced changes on mitochondrial protein expression in these cells and observe the distribution change in cytochrome C during apoptosis. The parallel detection by DHM, TIE and Raman spectroscopy allows us to monitor the dynamics of biomolecules with a temporal resolution of a few minutes, and possibly observe the oxidization state of intracellular molecules. Apoptosis is a rapid process. It is, therefore, a challenge to study the dynamics of protein-protein and protein-membrane interactions in apoptotic cells in-vivo, determine structural changes in membrane-bound proteins, and capture conformational changes in response to apoptotic stimuli in real time, all of which are key to understanding the mechanisms of both apoptosis induction and execution. The relationship between apoptosis and the cell cycle, and the paradoxical effect of some inducers of cell death in cell proliferation, has made it difficult to understand and identify apoptosis-specific regulatory mechanisms. Therefore, using advanced optics tools, such as QPI and Raman spectroscopy, to achieve important advances in molecular identification of the cell death machinery, is warranted. The proposed interdisciplinary project will employ a new imaging techniques to evaluate mitochondrial dynamics, which will advance both fundamental and applied aspects of biophysical research.